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Abstract:

The invention relates to kits and methods for assessing the
susceptibility of a human to oxidative stress or damage. The methods
involve assessing occurrence in the human's genome of one or more
polymorphisms (e.g., single nucleotide polymorphisms) that occur in one
or more genes associated with oxidative stress and that are associated
with a disorder in humans. Preferred assessment and scoring methods are
disclosed, as are kit for performing the methods.

Claims:

1. A method of assessing relative susceptibility of a human to oxidative
damage, the method comprising assessing occurrence in the human's genome
of disorder-associated polymorphisms in at least two genes selected from
the group consisting of a) genes which encode an enzyme that catalyzes
conversion of a toxic oxygen species to a less toxic oxygen species; b)
genes which encode a protein that provides protection against oxidative
stress; c) genes which encode a protein that induces production of a
toxic oxygen species; d) genes which encode a protein that indirectly
affects oxidative stress; and e) genes which encode a protein for which
the level of expression of the protein is associated with oxidative
stress, whereby occurrence of any of the polymorphisms is an indication
that the human is more susceptible to oxidative damage than a human whose
genome does not comprise the polymorphism, and whereby occurrence of a
plurality of the polymorphisms is an indication that the human is even
more susceptible to oxidative damage than a human whose genome does not
comprise the polymorphisms.

2.-109. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. patent application Ser.
No. 13/195,585, filed on Aug. 1, 2011, which is a Continuation of U.S.
patent application Ser. No. 11/931,447, filed on Oct. 31, 2007, which is
a Continuation of U.S. patent application Ser. No. 09/826,522, filed Apr.
5, 2001, (abandoned). All patents, patent applications, and references
cited in this application are hereby expressly incorporated by reference
herein in their entireties.

BACKGROUND OF THE INVENTION

[0002] Oxidation of the chemical components of foodstuffs provides energy
that is used to build and maintain the body and to enable normal
physiological function. Such oxidation involves a chain of chemical
reactions including reactions in which transfer of electrons from one
chemical compound to another are catalyzed. These reactions are catalyzed
by enzymes, which serve to align and chemically activate one or more of,
for example, reactants, cofactors, metal atoms or ions, and water
molecules. Despite the inherent specificity of enzyme-catalyzed
reactions, side reactions inevitably occur.

[0003] Oxygen is a common and relatively chemically reactive component of
biological systems. Diatomic oxygen is ordinarily relatively harmless to
body systems, as is fully reduced oxygen (i.e., water). However, transfer
of one or more electrons to oxygen (e.g., during reduction of oxygen to
water during oxidative phosphorylation or by way of a side reaction of
another biochemical process) can result in formation of more reactive
species of oxygen, such as hydrogen peroxide, superoxide radicals, and
hydroxyl radicals. These relatively reactive forms of oxygen can damage
biochemical components of the body such as proteins, lipids, and DNA,
destroying or inhibiting the normal function of the components.

[0004] The effects of biochemical damage inflicted by interaction of
reactive forms of oxygen with body components can be manifested in a
number of ways. DNA is the genetic material that carries the
`instructions` for making the components of a normal human body.
Oxidative damage to DNA can result in mutations (i.e., changes in the
`instructions`) that lead the body to make abnormal components. The
abnormal components can have inhibited (or no) ability to perform their
normal function, and this can be manifested as a disease or disorder.
Likewise, oxidative damage to enzymes or lipid components of membranes
can inhibit or ablate their normal function, and this too can be
manifested as a disease or disorder. The degree to which a cell or tissue
of a human body is subjected to damage caused by reactive forms of oxygen
is sometimes designated `oxidative stress.` The diseases and disorders
associated with oxidative damage to body components are thus
manifestations of oxidative stress. Aging is another manifestation of
oxidative stress. Over time, damage caused by interaction of reactive
forms of oxygen with body components degrades the structure and function
of those components, leading to detectable changes in body structure and
function.

[0005] If the human body were not able to detoxify reactive forms of
oxygen and mitigate their effects on the body, then human life would be
significantly shorter or even impossible. However, the human body
comprises enzymes which are able to catalyze transformation of reactive
forms of oxygen to less toxic species and other enzymes which are able to
repair damage done to body components by reactive forms of oxygen.

[0006] Most, if not all, human genes occur in a variety of forms which
differ in at least minor ways. Heterogeneity in human genes is believed
to have arisen, in part, from minor, non-fatal mutations that have
occurred in the genome over time. In some instances, differences between
alternative forms of a gene are manifested as differences in the amino
acid sequence of a protein encoded by the gene. Some amino acid sequence
differences can alter the reactivity or substrate specificity of the
protein. Differences between alternative forms of a gene can also affect
the degree to which (if at all) the gene is expressed. However, many
heterogeneities that occur in human genes appear not to be correlated
with any particular phenotype. Known heterogeneities include, for
example, single nucleotide polymorphisms (i.e., alternative forms of a
gene having a difference at a single nucleotide residue). Other known
polymorphic forms include those in which the sequence of larger (e.g.,
2-1000 residues) portions of a gene exhibits numerous sequence
differences and those which differ by the presence or absence of a
portion of a gene.

[0007] Numerous disorders and physiological states have been correlated
with occurrence of one or more alternative forms of a gene in the genome
of a human who exhibits the disorder or physiological state. For example,
Kimura et al. (2000, Am. J. Ophthalmol. 130:769-773) discloses an
association between occurrence of a SNP of the manganese superoxide
dismutase gene and a form of macular degeneration. Although associations
between individual disorders and individual genetic polymorphisms are
known, a need remains for a method of assessing the overall state of
oxidative stress to which a human is subjected. The invention satisfies
this need.

BRIEF SUMMARY OF THE INVENTION

[0008] The invention relates to a method of assessing relative
susceptibility of a human to oxidative damage. The method comprises
assessing occurrence in the human's genome of disorder-associated
polymorphisms (e.g., single nucleotide polymorphisms; SNPs) in at least
two (and preferably three, four, six, ten, fifteen, or twenty or more)
genes selected from the group consisting of

[0009] a) genes which encode an enzyme that catalyzes conversion of a
toxic oxygen species to a less toxic oxygen species;

[0010] b) genes which encode a protein that provides protection against
oxidative stress;

[0011] c) genes which encode a protein that induces production of a toxic
oxygen species;

[0012] d) genes which encode a protein that indirectly affects oxidative
stress; and

[0013] e) genes which encode a protein for which the level of expression
of the protein is associated with oxidative stress.

[0014] Occurrence of any of the polymorphisms is an indication that the
human is more susceptible to oxidative damage than a human whose genome
does not comprise the polymorphism. Furthermore, occurrence of a
plurality of the polymorphisms is an indication that the human is even
more susceptible to oxidative damage than a human whose genome does not
comprise the polymorphisms. Preferably the genes are selected from the
group consisting of a), b), c), and d), and more preferably they are
selected from the group consisting of a), b), and c). In one embodiment,
the method comprises assessing occurrence in the human's genome of
disorder-associated polymorphisms in at least four genes selected from
the group consisting of genes which encode an enzyme that catalyzes
conversion of a toxic oxygen species to a less toxic oxygen species
(e.g., genes which encode mitochondrial manganese superoxide dismutase,
cytoplasmic copper/zinc superoxide dismutase, catalase, and glutathione
peroxidase).

[0015] The method by which occurrence of an individual disorder-associated
polymorphism is assessed is not critical. For example, occurrence of the
polymorphisms can be assessed using a method that includes contacting a
nucleic acid derived from the human's genome with a first
oligonucleotide. The first oligonucleotide can be one that anneals with
higher stringency with the disorder-associated polymorphism than with a
corresponding non-disorder-associated polymorphism. Annealing of the
first oligonucleotide and the nucleic acid can be assessed, and such
annealing is an indication that the human's genome comprises the
disorder-associated polymorphism. Use of an oligonucleotide has the
advantage that the oligonucleotide can be attached to a support using
routine methods, and that a plurality of oligonucleotides can be attached
to the same support, to allow simultaneous detection of multiple
polymorphisms. If a second oligonucleotide which anneals with higher
stringency with a non-disorder-associated polymorphism than with a
corresponding disorder-associated polymorphism is used, then the allelic
content of the human's genome can be determined. Detection of polymorphic
sequences can be simplified by using labeled oligonucleotides, such as
molecular beacon oligonucleotides.

[0016] Once the content of the human's genome for disorder-associated
polymorphisms has been assessed, assessment of susceptibility to
oxidative damage can further comprise calculating a susceptibility score
for the human. A susceptibility score can be calculated by summing, for
each of the selected genes in which a disorder-associated polymorphism
occurs in the human's genome, the product of a constant and a correlation
factor. The correlation factor can, alternatively, be a factor that
represents the fraction of humans heterozygous for the
disorder-associated polymorphism who exhibit the corresponding disorder
or a factor that represents the fraction of humans homozygous for the
disorder-associated polymorphism who exhibit the corresponding disorder.
The constant can be selected based on the known or surmised relevance of
the gene with respect to oxidative damage. The susceptibility score
represents the relative susceptibility of the human to oxidative damage.

[0017] In another aspect, the invention relates to a method of selecting a
dose of an anti-oxidant composition (i.e., a composition comprising a
compound that exhibits anti-oxidant properties, such as vitamin E or
vitamin C, or a compound that can otherwise supplement the body's normal
anti-oxidant mechanisms, such as alpha-lipoic acid and coenzyme Q) for
administration to a human. This method comprises assessing occurrence in
the human's genome of disorder-associated polymorphisms in at least one
of the genes selected from the group consisting of a), b), c), d), and
e), as indicated above. After assessing occurrence of the polymorphisms,
a dose of the composition is selected. Occurrence of any of the
polymorphisms is an indication that a greater dose of the composition
should be administered to the human.

[0018] The invention also relates to a kit for assessing relative
susceptibility of a human to oxidative damage. The kit comprises reagents
for assessing occurrence in the human's genome of disorder-associated
polymorphisms in at least one gene selected from the group consisting of
a), b), c), d), and e), as indicated above. Examples of suitable reagents
include oligonucleotides (e.g., molecular beacon oligonucleotides) that
anneal with higher stringency with the disorder-associated polymorphisms
than with corresponding non-disorder-associated polymorphisms and
oligonucleotide primers that are complementary to the region adjacent a
characteristic residue of the disorder-associated polymorphism. These
primers are useful for amplifying at least the characteristic residue,
thereby facilitating its detection. The kit can further comprise an
instructional material which includes a numerical value representing the
product of a constant and a correlation factor.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0019] The foregoing summary, as well as the following detailed
description of preferred embodiments of the invention, will be better
understood when read in conjunction with the appended drawings. The
invention is not limited to the precise arrangements and
instrumentalities shown.

[0020] FIGS. 1A and 1B are images which depict examples of results that
can be obtained by analyzing occurrence of polymorphisms in several
genes. The results shown in FIG. 1A are derived from a hypothetical first
human, and those shown in FIG. 1B are derived from a hypothetical second
human. Circles represent different polymorphisms of the gene indicated to
the left of the row of circles. Filled circles indicate the presence of
the polymorphism. Non-filled circles indicate the absence of the
polymorphism. Numbers below each circle represent a correlation factor
for the polymorphism and a disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention relates to kits and methods for assessing the
relative susceptibility of a human to oxidative damage by assessing
occurrence in the human's genome of genetic polymorphisms that are
associated with disorders. Crudely simplified, the methods involve
determining whether one or more polymorphisms that have been associated
(by the inventors or by others) with a disorder (e.g., a disease or
pathological state) in humans occur in the genome of the human being
tested. In some embodiments, the number of polymorphisms that occur in
the human's genome are summed to yield a value; the higher the value is,
the greater the susceptibility of the human to oxidative damage is
assessed to be. In other embodiments, a weighting factor is assigned to
each polymorphism tested, and the weighting factors of polymorphisms that
occur in the human's genome are summed to yield a value that represents
relative susceptibility to oxidative damage. The weighting factor can
represent the product of a constant assigned to the gene in which the
corresponding polymorphism occurs and a correlation factor that describes
how informative occurrence of the polymorphism is for occurrence of the
disorder with which it is associated. The invention includes a variety of
alternative methods and kits for performing the methods, as described in
greater detail herein.

DEFINITIONS

[0022] As used in this disclosure, the following terms have the meanings
associated with them in this section.

[0023] A "polymorphism" in a gene is one of the alternative fonns of a
portion of the gene that are known to occur in the human population. For
example, many genes are known to exhibit single nucleotide polymorphic
forms, whereby the identity of a single nucleotide residue of the gene
differs among the forms. Each of the polymorphic forms represents a
single polymorphism, as the term is used herein. Other known polymorphic
forms include alternative forms in which multiple consecutive or
closely-spaced, non-consecutive nucleotide residues vary in sequence,
forms which differ by the presence or absence of a single nucleotide
residue or a small number of nucleotide residues, and forms which exhibit
different mRNA splicing patterns.

[0024] A "single nucleotide polymorphism" ("SNP") is one of the
alternative forms of a portion of a gene that vary only in the identity
of a single nucleotide residue in that portion.

[0025] A "disorder-associated" polymorphism is an alternative form of a
portion of a gene, wherein occurrence of the alternative form in the
genome of a human has been correlated with exhibition by the human of a
disease or a pathological state.

[0026] A "non-disorder-associated" polymorphism is an alternative form of
a portion of a gene for which no significant correlation has been made
between occurrence of the alternative form in the genome and a disease or
a pathological state. Non-disorder-associated polymorphisms are sometimes
designated "neutral" polymorphisms in the art.

[0027] A disorder-associated polymorphism and a non-disease-associated
polymorphism "correspond" with one another if the two polymorphisms are
two alternative forms of the same portion of the gene. By way of example,
if the identity of residue 100 of a gene is adenine in a
disorder-associated polymorphism of the gene and cytosine in a
non-disorder-associated polymorphism of the gene, then the two
polymorphisms correspond with one another. It is understood that there
may be three or more corresponding polymorphisms when there are more than
two alternative forms of the same portion of the gene.

[0028] A "characteristic residue" of a polymorphism is a nucleotide
residue, the identity of which is known to vary among the alternative
forms corresponding to the polymorphism.

[0029] "Toxic oxygen species" include, in approximate order of reactivity,
hydroxyl radicals, superoxide radicals, nitric oxide, peroxy nitrite
(ONOO.sup.-; the product of a reaction between nitric oxide and
superoxide radical), and hydrogen peroxide. Ordinary diatomic oxygen is
not a toxic oxygen species, as the term is used herein.

[0030] "Oxidative damage" refers to chemical reaction of a normal cellular
component (e.g., DNA, a protein, or a lipid) with a toxic oxygen species,
whereby at least one normal function of the component is inhibited or
eliminated. The terms "oxidative damage" and "oxidative stress" are used
interchangeably herein.

[0031] A "molecular beacon oligonucleotide" is a single-stranded
oligonucleotide having a fluorescent label (e.g., rhodamine, FAM, TET,
VIC, JOE, or HEX) attached to the 5'-end thereof and a fluorescence
quencher (e.g., TAMRA or DABCYL) attached to the 3'-end thereof (or vice
versa), as described (Kostrikis et al., 1998, Science 279:1228-1229).

[0032] Two molecular beacon oligonucleotides are "spectrally distinct" if
they can be differentially detected using spectrophotometric or
spectrofluorimetric methods. Examples of characteristics that can be used
to differentiate spectrally distinct oligonucleotides include absorption
or excitation wavelength, emission wavelength, and fluorescent lifetime.

[0033] An "instructional material" is a publication, a recording, a
diagram, or any other medium of expression which can be used to
communicate how to use a kit described herein, numerical values for
weighting the significance of various polymorphisms that are detectable
using the kit, or both. The instructional material of the kit of the
invention can, for example, be affixed to a container which contains a
kit of the invention or be shipped together with a container which
contains the kit. Alternatively, the instructional material can be
shipped separately from the container with the intention that the
instructional material and the kit be used cooperatively by the
recipient.

[0034] The "stringency" with which two polynucleotides anneal means the
relative likelihood that the polynucleotides will anneal in a solution as
the conditions of the solution become less favorable for annealing.
Examples of stringent conditions are known in the art and can be found in
available references (e.g., Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous annealing
methods are described in that reference and either can be used. In
general, a first pair of polynucleotides anneal with higher stringency
than a second pair if the first pair is more likely to anneal (or remain
annealed) as one or more of the salt concentration, temperature, and
detergent concentration are increased.

[0035] With respect to a disorder, a "correlation factor" for a
disorder-associated polymorphism is the fraction of humans who are
heterozygous or homozygous for the polymorphism who exhibit the disorder.
The correlation factor can, alternatively, be based solely on those who
are heterozygous, solely on those who are homozygous, or on those who are
either heterozygous or homozygous,

[0036] A "non-extendable" nucleotide residue is a nucleotide residue that
is capable of being added to a polynucleotide by a polymerase (i.e., by
extension of the polynucleotide in association with a complement thereof,
catalyzed by the polymerase) and that, upon addition to the
polynucleotide, renders the polynucleotide incapable of being further
extended by the polymerase.

DESCRIPTION

[0037] The invention relates to kits and methods for assessing the
relative susceptibility of a human to oxidative damage by assessing
occurrence in the human's genome of genetic polymorphisms that are
associated with disorders.

[0038] It has been discovered that the degree to which a human is
susceptible to oxidative damage can be assessed by determining which
polymorphic forms of certain genes are present in the human's genome. The
genes which are assessed are genes that are associated with oxidative
stress, including both genes which provide protection against oxidative
damage and genes which exacerbate oxidative damage.

[0039] Among the types of genes which protect the body against oxidative
stress are genes which encode an enzyme that catalyzes conversion of a
toxic oxygen species to a less toxic oxygen species, genes that encode a
protein that directly provides protection against oxidative damage, and
genes which encode a protein that indirectly provides protection against
oxidative damage.

[0040] Among enzymes that catalyze conversion of a toxic oxygen species to
a less toxic oxygen species, four are of particular relevance, namely
mitochondrial manganese superoxide dismutase (MnSOD), cytoplasmic
copper/zinc superoxide dismutase (CZSOD), catalase (CAT), and glutathione
peroxidase (GP). Polymorphisms that occur in these genes are known to be
associated with various disorders (see, e.g., Kimura et al., 2000, Am. J.
Ophthalmol. 130:769-773). Occurrence of disorder-associated polymorphisms
in at least one (and preferably two, three, or all) of these four genes
should be assessed in the methods described herein, given the importance
of these genes. Similarly, the kits described herein preferably include
reagents for detecting disorder-associated polymorphisms in at least one
(and preferably two, three, or all) of these four genes. In addition, the
significance of occurrence of disorder-associated polymorphisms in these
genes can be applied by assigning a greater weighting factor to
disorder-associated polymorphisms of these genes than to
disorder-associated polymorphisms in other genes associated with
oxidative stress.

[0041] It was not previously appreciated that detection in a human's
genome of two or more disorder-associated polymorphisms in genes
associated with oxidative stress is indicative that the human globally
exhibits enhanced susceptibility to oxidative damage. Previous studies
are believed to have recognized only association between a polymorphism
in one of these genes and a particular disorder (e.g., exudative macular
degeneration in the Kimura reference). The inventors believe that they
are the first to describe methods and kits for assessing a human's global
(i.e., not limited to a particular tissue, cell type, or organ)
susceptibility to oxidative damage.

[0042] In addition to the MnSOD, CZSOD, CAT, and GP genes mentioned above,
other genes encode proteins which provide direct or indirect protection
against oxidative damage, for example by converting toxic species of
oxygen to less toxic species, by eliminating precursors of toxic forms of
oxygen, or by repairing oxidative damage. Examples of these genes include
those which encode glutathione S-transferase, glutathione reductase,
thioredoxin reductase, paraoxonase, NAD(P)H:quinone oxidoreductases 1 and
2, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase, and epoxide hydrolase.
Detection in a human genome of disorder-associated polymorphisms in one
or more of these genes indicates that the human exhibits enhanced
susceptibility to oxidative damage. The methods and kits described herein
can use this indication to assess the susceptibility of a human to
oxidative stress.

[0043] Among the genes which exacerbate oxidative damage are genes which
encode a protein that induces production of a toxic oxygen species,
either directly (e.g., by catalyzing a reaction in which a toxic species
of oxygen is a direct or side product) or indirectly (e.g., by enhancing
flux through a metabolic pathway that leads to production of a toxic
species of oxygen). Examples of proteins that directly or indirectly
induce production of toxic oxygen species include myeloperoxidase, tumor
necrosis factor alpha, NADH/NADPH oxidase p22 phox protein, nitric oxide
synthase xanthine oxidase, and cytochrome P450. Detection in a human
genome of disorder-associated polymorphisms in one or more genes encoding
one of these proteins indicates that the human exhibits enhanced
susceptibility to oxidative damage.

[0044] The methods described herein can also be used to assess
susceptibility to oxidative damage by determining the presence in a
human's genome of polymorphic forms of genes that are associated with
oxidative damage, regardless of whether the mechanism by which the gene
affects oxidative stress is understood. By way of example, apolipoprotein
E is a multi-functional molecule that is able to affect oxidative stress.
The ApoE4 phenotype, for example, is known to be associated with enhanced
hydroxyl radical levels in patients afflicted with Alzheimer's disease,
and ApoE expression is known to exacerbate oxidative stress. Further by
way of example, enhancement of oxidative stress is known to be associated
with each of elevated homocysteine level, depressed serum bilirubin
level, depressed acid phosphatase activity, depressed protein
phosphotyrosine phosphatase activity, and depressed epinephrine oxidase
activity. Thus, occurrence in the genome of polymorphisms in genes which
encode proteins that affect these levels and activities can be
determined, and their occurrence can be used to estimate susceptibility
of the human to oxidative stress. Examples of genes for which
polymorphisms can be associated with altered susceptibility to oxidative
damage include UDP-glucuronosyltransferase 1A (i.e., the UGT1A1 gene),
genes encoding acid phosphatase, protein phosphotyrosine phosphatase,
epinephrine oxidase, ApoE4, cystathionine beta-synthase, cystathionine
gamma-lyase, N5-methyl THF:homocysteine methyltransferase, and
S-adenosylmethionine methyltransferase.

[0045] Heat shock proteins are also known to provide at least indirect
protection of cells from oxidative damage, and occurrence of a heat shock
protein gene polymorphism can be used as informative markers of
susceptibility to oxidative damage when the polymorphism is known to be a
disorder-associated polymorphism.

[0046] Examples of the polymorphisms in the foregoing genes which can be
informative for susceptibility to oxidative damage include the following:
[0047] a polymorphism manifested as a change from an alanine residue to
a valine residue at amino acid residue 9 (i.e., in the signal sequence)
of MnSOD; [0048] a polymorphism manifested as a change from an isoleucine
residue to a thymine residue at amino acid residue 58 of MnSOD; [0049] a
polymorphism manifested as a change from a valine residue to a glutamic
acid residue at amino acid residue 7 of CZSOD; [0050] a polymorphism
manifested as a change from a cysteine residue to a phenylalanine residue
at amino acid residue 6 of CZSOD; [0051] a polymorphism manifested as a
change from a cytosine residue to a thymine residue at nucleotide residue
-262 (i.e., in the promoter region) of the catalase gene; [0052] a
polymorphism in the hGPX1 gene manifested as a change from a proline
residue to a leucine residue at amino acid residue 198 of glutathione
peroxidase; [0053] a polymorphism in the GSTP1 gene manifested as a
change from a valine residue to an isoleucine residue at amino acid
residue 105 of glutathione peroxidase; [0054] a polymorphism manifested
as a change from a thymine residue to a cytosine residue at nucleotide
residue -107 (i.e., in the promoter region) of the gene which encodes
paraoxonase; [0055] a polymorphism manifested as a change from a cytosine
residue to a thymine residue at nucleotide residue 242 (i.e., in the
coding region) of the gene encoding NAD(P)H:quinone oxidoreductase;
[0056] a polymorphism manifested as a change from a thymine residue to a
cytosine residue at nucleotide residue 113 in exon 3 of the gene which
encodes epoxide hydrolase (i.e., effecting change from a tyrosine residue
to a histidine residue in epoxide hydrolase); [0057] a polymorphism
manifested as a change from a guanine residue to an adenine residue at
nucleotide residue -463 (i.e., in the promoter region) of the gene which
encodes myeloperoxidase; [0058] a polymorphism manifested as a change to
an adenine residue at nucleotide residue -238 (i.e., in the promoter
region) of the gene which encodes tumor necrosis factor alpha (i.e., the
TNF promoter variant designated TNF2); [0059] a polymorphism manifested
as a change to an adenine residue at nucleotide residue -308 (i.e., in
the promoter region) of the gene which encodes tumor necrosis factor
alpha (i.e., the TNF promoter variant designated TNF3); [0060] a
polymorphism manifested as a change from a cytosine residue to a thymine
residue at nucleotide residue 242 (i.e., in the coding region) of the
phox gene encoding the NADH/NADPH oxidase p22 subunit; [0061] a
polymorphism manifested as a 27 base pair repeat in intron 4 (i.e.,
between nucleotide residues 5130 and 5511) of the gene encoding nitric
oxide synthase; [0062] a polymorphism manifested as a change from an
adenine residue to a guanine residue at nucleotide residue -290 (i.e., in
the 5'-flanking region) of the gene encoding cytochrome P450 (i.e., the
polymorphism designated the CYP3A4 cytochrome P450 variant); the
polymorphism designated the ApoE4 allele of the ApoE gene; and [0063] a
polymorphism manifested as a change from a cytosine residue to a thymine
residue at nucleotide residue 699 (i.e., in the coding region) of the
gene encoding cystathionine beta-synthase.

Methods of Assessing Susceptibility to Oxidative Damage

[0064] The invention includes a method of assessing the relative
susceptibility of a human to oxidative damage. This susceptibility can be
calculated relative to a hypothetical human whose genome does not contain
a single disorder-associated polymorphism in a gene associated with
oxidative stress. Alternatively, susceptibility can be calculated
relative to another human who may have one or more different
disorder-associated polymorphism than the human being assessed. In
practice, the basis upon which raw susceptibility scores are calculated
is immaterial, so long as the same basis is used for all humans whose
scores are to be compared (i.e., so that the scores are relatable to one
another).

[0065] The relative susceptibility of a human to oxidative damage permits
assessment of risks and benefits of a variety of compositions,
conditions, and interventions. In one embodiment, the susceptibility of a
human to oxidative damage can be used to determine whether the human
would benefit by supplementing nutritional intake with a composition that
contains one or more anti-oxidants. Furthermore, the relative
susceptibility of the human to oxidative damage can indicate an
appropriate dose of such an anti-oxidant-containing composition. In
another embodiment, suitability of a condition or intervention for a
human (e.g., administration to the human of hyperbaric oxygen or a
pharmaceutical agent known to induce generation of toxic species of
oxygen) can be determined by assessing the human's susceptibility to
oxidative damage.

[0066] Susceptibility of a human to oxidative damage is assessed by
assessing occurrence in the human's genome of disorder-associated
polymorphisms in a plurality of genes associated with oxidative stress
(e.g., 3, 4, 6, 8, 10, 15, 20, or 30 or more genes). Occurrence of a
disorder-associated polymorphism in one of these genes is an indication
that the human has a greater susceptibility to oxidative damage than a
human in whose genome the polymorphism does not occur. Of course,
occurrence of two or more such polymorphisms in the human's genome
indicates that the human exhibits even greater susceptibility to
oxidative damages.

[0067] Occurrence of every disorder-associated polymorphism in a gene
related to oxidative stress is not necessarily equally indicative of
susceptibility to oxidative stress. In order to account for differences
in the significance of various disorder-associated polymorphisms, a
weighting factor can be assigned to each polymorphism detected in the
methods and kits described herein. As indicated above, four genes (MnSOD,
CZSOD, CAT, and GP) are known to have very significant roles in oxidative
stress in humans. All else being equal, disorder-associated polymorphisms
that occur in one of these four genes are more significant than
polymorphisms that occur in genes having less significant roles in
oxidative stress. Thus, a greater weighting factor can be assigned to
these polymorphisms than to others. By way of example, the weighting
factor assigned to these four polymorphisms can be 1 to 10 times greater
than the weighting factor assigned to disorder-associated polymorphisms
(having equal correlation with the corresponding disorder, as discussed
below) in other genes. Preferably, the weighting factor assigned to
polymorphisms in the MnSOD, CZSOD, CAT, and GP genes is twice that
assigned to disorder-associated polymorphisms in other genes.

[0068] Another factor which can influence the significance that is
assigned to occurrence of a disorder-associated polymorphism in a human's
genome is the degree to which the polymorphism is correlated with the
corresponding disorder. Some disorders are highly correlated with
occurrence of a genetic polymorphism, and other disorders exhibit lower
correlation with a polymorphism. When a polymorphism is reported to be
associated with a disorder (i.e., with a disease or pathological
condition), a degree of correlation between the polymorphism and the
disorder is often reported. One useful way of calculating a factor that
describes correlation between a polymorphism and a disorder is to
calculate an odds ratio that describes the likelihood that an individual
in whose genome the disorder-associate polymorphism occurs will exhibit
or develop the disorder. Because the kits and methods described herein
can be used to detect whether the human is homozygous for the
disease-associated polymorphism, odds ratios calculated for homozygous
individuals can also be used, if they are available. Odds ratios can be
calculated as described in the art.

[0069] For a disorder-associated polymorphism, the odds ratio can be
calculated as follows. First, the odds of being afflicted with the
disorder are calculated for a first population in whom the polymorphism
occurs by dividing the number of afflicted individuals in the first
population by the total number of individuals in the first population.
Second, the odds of being afflicted with the disorder are calculated for
a first population in whom the polymorphism does not occur by dividing
the number of afflicted individuals in the second population by the total
number of individuals in the second population. Third, the odds ratio is
calculated by dividing the odds for the first population by the odds for
the second population. If the odds ratio is greater than one, then this
is an indication that occurrence of the polymorphism is associated with
occurrence of the disorder. Furthermore, the magnitude of the odds ratio
is an indication of the significance of the association.

[0070] An overall oxidative stress susceptibility score for a human can be
determined as follows. A significance score can be assigned to each
disorder-associated polymorphism that is detected in the human's genome
using a method or kit described herein. The significance score is a
constant (e.g., 1.00), and is multiplied by any significance factor
(e.g., 1-10, preferably 2, for the MnSOD, CZSOD, CAT, and GP genes) and
by any correlation factor that is available. If information is available
which describes the correlation between homozygosity for the polymorphism
and the corresponding disorder, then that correlation factor should be
used in place of the correlation factor for mere occurrence of the
polymorphism, at least if the method or kit is used to rule out
occurrence in the subject's genome of corresponding
non-disorder-associated polymorphisms. If significance and correlation
factors are not available, then values of 1.00 should be assigned to
each. An overall score is determined by summing the significance score
for each disorder-associated polymorphism that is detected using the
method or kit. This overall oxidative stress susceptibility score can be
compared with the values obtained from other subjects, or it can be
compared with the value (i.e., zero) which would be expected to occur in
a human whose genome does not include any disorder-associated
polymorphism in a gene associated with oxidative stress.

[0071] By way of example, the Kimura reference describes two corresponding
polymorphisms that occur in the MnSOD gene (i.e., occurrence of either C
or T at a particular position in the MnSOD gene). Individuals in whose
genome the disorder-associated polymorphism occur exhibit an odds ratio
of 1.43 for the disorder (a form of macular degeneration), and
individuals who are homozygous for the same polymorphism exhibit an odds
ratio of 10.14. Thus, when the MnSOD gene is one of the genes assessed in
the methods and kits described herein, a weighting factor of 1.43 can be
applied to occurrence of this disorder-associated polymorphism in the
subject's genome, and a weighting factor of 10,14 can be applied if the
method or kit is used to determine that no other corresponding
polymorphism occurs in the subject's genome. As indicated herein, an
additional factor can be combined with this factor to represent the
significance of the MnSOD gene in oxidative stress. Thus, if this latter
factor is selected to be 2, then occurrence of the disorder-associated
polymorphism described in Kimura can be assigned a significance of 2.86,
and exclusive occurrence of that polymorphism (i.e., homozygosity) can be
assigned a significance of 20.28.

[0072] The method used to assess occurrence of any particular
disorder-associated polymorphism (or non-disorder-associated
polymorphism) is not critical. Numerous methods of detecting occurrence
of a polymorphism are known in the art, and substantially any of those
methods can be used in the kits and methods described herein. Naturally,
the reagents included in the kit will vary depending on the method to be
used to detect the polymorphisms. Examples of some suitable polymorphism
detection methods are provided below.

[0073] In one embodiment, a pair of oligonucleotide primers are used to
amplify a portion of the gene that includes a polymorphic region.
Detection of one or more of the polymorphisms that occur at the
polymorphic region can be achieved by contacting the amplified portion
with an oligonucleotide having a sequence that will anneal under
stringent conditions with the amplified portion only if one polymorphism
occurs at the portion, but will not anneal with the amplified portion if
another polymorphism occurs at that portion. Various acceptable stringent
conditions are known in the art, and can be modified by the skilled
artisan as appropriate to any particular amplified
portion/oligonucleotide pair. An example of stringent conditions is
hybridization in 6× sodium chloride/sodium citrate (SSC) at about
45° C., followed by one or more washes in 0.2×SSC, 0.1%
(w/v) SDS at 50° C.

[0074] In an alternative embodiment, one or more molecular beacon
oligonucleotides are used to detect polymorphisms (disorder-associated,
non-disorder-associated, or both) in a sample that contains a copy of the
subject's genome, a fraction of the subject's genome, or amplification
products generated from the subject's genome (e.g., amplified portions of
oxidative stress-associated genes in which portions polymorphisms are
known to occur).

[0075] Molecular beacon probes are single-stranded oligonucleotides having
a fluorescent label (e.g. rhodamine, FAM, TET, VIC, JOE, or HEX) attached
to the 5'-end thereof and a fluorescence quencher (e.g. TAMRA or DABCYL)
attached to the 3'-end thereof (or vice versa), as described (Kostrikis
et al., 1998, Science 279:1228-1229). The sequence of each molecular
beacon probe is selected to include two complementary hairpin regions,
whereby the probe can self-anneal to form a hairpin structure. The 5'-
and 3'-ends are brought into close association when the hairpin structure
forms. The probe also comprises a targeting portion which is selected to
be complementary to a target sequence (e.g. a single polymorphism of an
oxidative-stress-associated gene). The targeting portion and at least one
of the hairpin regions are located in close proximity to one another,
meaning that the targeting portion either overlaps the hairpin region or
flanks it, having no more than about 5 nucleotide residues therebetween.

[0076] If the hairpin regions of the molecular beacon probe anneal with
one another, then the probe does not fluoresce, because the hairpin
structure forms and the fluorescence quencher attached to one end of the
probe quenches fluorescence of the label attached to the other end of the
probe. If the targeting portion of the probe anneals with a region of a
nucleic acid having the target sequence, then formation of the hairpin
structure is inhibited, the fluorescence quencher is not brought into
association with the fluorescent label, and the probe fluoresces.
Multiple molecular beacon probes can be used in a single reaction
mixture, and fluorescence associated with the probes can be
differentiated if the molecular beacon probes are spectrally distinct.

[0077] Thus, in this embodiment, one or more molecular beacon probes are
used, each having a targeting portion which is complementary to a target
region (e.g. 20 to 40 nucleotide residues, more preferably 20 to 30
residues) of one polymorphism of an oxidative stress-associated gene
(e.g., one of the genes disclosed herein). If the polymorphism to be
detected is a single nucleotide polymorphism (SNP), then the target
region includes, and preferably is approximately centered around, the
nucleotide residue at which the polymorphism occurs. More preferably, two
such probes are used, one having a targeting region completely
complementary to the target region of one polymorphism of the gene (e.g.,
one of two polymorphisms of an SNP), and the other having a targeting
region completely complementary to the target region of a corresponding
polymorphism of the gene (e.g., the other polymorphism of the SNP).

[0078] In yet another embodiment of how polymorphisms in an oxidative
damage associated gene can be assessed, oligonucleotide primers which are
complementary to a region adjacent a characteristic residue of the
polymorphism are extended using a polymerase enzyme, and the identity of
the nucleotide residue that is added to the primer in the position
complementary to the characteristic residue is determined. The primer can
be extended in the presence of non-extendable nucleotide residues in
order to ensure that a limited number of (or only one) nucleotide
residues are incorporated into the primer. Methods of this type are known
in the art (e.g., the SNP-ITS technology of Orchid Biocomputer, Inc.) and
are described, for example in U.S. Pat. Nos. 6,013,431 and 6,004,744.

Kits for Assessing Oxidative Stress

[0079] The invention includes a kit for assessing the relative
susceptibility of a human to oxidative stress. The kit contains reagents
for performing one or more of the methods described herein. The reagents
used in certain embodiments of the methods described herein are indicated
above. Reagents useful for performing those methods using a variety of
alternative sample preparation and polymorphism detection methods or
chemistries are apparent to the skilled artisan.

[0080] Kits for detecting polymorphisms in individual genes are known in
the art, and the kit of the invention can have similar components.
However, a critical feature of the kit is that it includes reagents that
permit its user to detect disorder-associated polymorphisms in at least
three genes associated with oxidative stress. Preferably the kit includes
reagents that permit detection of disorder-associated polymorphisms in at
least 4, 6, 8, 10, 15, 20, or 30 or more such genes.

[0081] In one embodiment, the kit includes a plurality of oligonucleotides
which anneal under stringent conditions with a disorder-associated
polymorphism of one of the genes, but not with a non-disorder
associated-polymorphism. Each of the oligonucleotides is preferably
attached to a surface in order to facilitate handling of the
oligonucleotide. The oligonucleotides can be linked with a plurality of
surfaces (e.g., oligonucleotides for a particular polymorphism being
attached to a particle discrete from a particle to which oligonucleotides
for another polymorphism are attached), or they can be attached to
discrete regions of a single surface (e.g., as in the GENECHIP® device
of Affymetrix, Inc.). Annealing between individual oligonucleotides and
the polymorphism corresponding thereto can be detected using standard
methods. The kit can also comprise oligonucleotides that are useful as
molecular beacon probes or as extendable primers.

[0082] In one embodiment, the kit further comprises a DNA collection kit
or apparatus, such as that described in co-pending U.S. patent
application Ser. No. 09/302,623 (allowed). Advantageously, DNA collected
using the kit or apparatus can be stored or archived, and subjected to
additional testing as previously unknown polymorphisms are discovered in
genes associated with oxidative stress, or as the significance of
previously unappreciated polymorphisms is realized.

[0083] It will be appreciated by those skilled in the art that changes can
made to the embodiments described above without departing from the broad
inventive concept thereof.

[0084] This invention is not limited to the particular embodiments
disclosed, and includes modifications within the spirit and scope of the
present invention as defined by the appended claims.